阅读说明：本技术 一种用于新型冠状病毒对结肠细胞影响机制研究的微流控结肠细胞研究模型及方法 (Microfluidic colon cell research model and method for researching influence mechanism of novel coronavirus on colon cells ) 是由 张莹 关茗洋 陈凤格 赵川 范尉尉 马辉 于 2021-05-21 设计创作，主要内容包括：本发明属体外模拟分析技术领域,具体涉及一种用于新型冠状病毒对结肠细胞影响机制研究的微流控结肠细胞研究模型,包括多于一个培养机构,选取其中一个所述培养机构作为对照组,其他所述培养机构作为实验组；所述培养机构包括培养池、多于一个收集池；所述收集池入口与所述培养池出口通过液体通道阀相连。有益效果在于：模拟装置结构简单,成本低；分别设置了A549细胞层以及人结肠杯状细胞HT29-CL.16E或HUVEC细胞层,有效模拟了结肠毛细血管的结构；利用真空泵的节律性的拉伸来模拟人体呼吸引起的结肠收缩和舒张；模拟装置操作简单；交换腔采用流线纺锤形,考虑到流体力学的因素,防止流体流动不均匀。(The invention belongs to the technical field of in-vitro simulation analysis, and particularly relates to a microfluidic colon cell research model for researching the influence mechanism of a novel coronavirus on colon cells, which comprises more than one culture mechanism, wherein one culture mechanism is selected as a control group, and other culture mechanisms are selected as experimental groups; the culture mechanism comprises a culture pond and more than one collecting pond; and the inlet of the collecting tank is connected with the outlet of the culture tank through a liquid channel valve. Has the advantages that: the simulation device has simple structure and low cost; an A549 cell layer and a human colon goblet cell HT29-CL.16E or HUVEC cell layer are respectively arranged, so that the structure of colon capillaries is effectively simulated; the rhythmic stretching of the vacuum pump is utilized to simulate the contraction and relaxation of the colon caused by the respiration of the human body; the simulation device is simple to operate; the exchange cavity adopts a streamline spindle shape, and the fluid flow is prevented from being uneven by considering the factor of fluid mechanics.)

1. A micro-fluidic colon cell research model for researching the influence mechanism of a novel coronavirus on colon cells is characterized by comprising more than one culture mechanism (1), wherein one culture mechanism (1) is selected as a control group, and the other culture mechanisms (1) are selected as experimental groups; the culture mechanism (1) comprises a culture pond (11) and more than one collecting pond (12); the inlet of the collecting pool (12) is connected with the outlet of the culture pool (11) through a liquid channel valve;

the culture pond (11) comprises a simulated outer cavity (111), a barrier layer (112), a simulated inner cavity (113) and a circulating passage (114); the barrier layer (112) is arranged between the simulated outer cavity (111) and the simulated inner cavity (113); the simulated outer cavity (111) and the simulated inner cavity (113) are respectively provided with the circulating passage (114); the barrier layer (112) comprises a skeleton layer (1121), a first simulation layer (1122), a second simulation layer (1123); the first mimic layer (1122) is a layer of HUVEC cells that proliferate on a first surface of the scaffold layer (1121); the second simulated layer (1123) is a layer of A549 cells proliferated on a second surface of the scaffold layer (1121); the first simulation layer (1122) is connected to the simulated external cavity (111) and the second simulation layer (1123) is connected to the simulated internal cavity (113).

2. The microfluidic colon cell research model for the research on the influence mechanism of the novel coronavirus on the colon cells as claimed in claim 1, wherein the inlet of the culture tank (11) adopts a serpentine path.

3. The microfluidic colon cell research model for researching the influence mechanism of the novel coronavirus on the colon cells as claimed in claim 1, wherein the framework layer (1121) is provided with holes, and the simulation layer covers the surface of the framework layer (1121) and simultaneously blocks the holes; or the framework layer (1121) adopts a semipermeable membrane, and substances on two sides of the barrier layer (112) can exchange substances with the simulation layer through the framework layer (1121).

4. The microfluidic colon cell research model for research on the influence mechanism of the novel coronavirus on the colon cells as claimed in claim 1, wherein the framework layer (1121) is made of polycarbonate.

5. The microfluidic colon cell study model for the study of the influence mechanism of novel coronaviruses on colon cells of claim 1, wherein the first simulation layer (1122) employs human colon goblet cells HT29-CL.16E or HUVEC with a cell density of 1 x 10⁷Per mL; the second simulated layer (1123) employs an A549 cell density of 1 × 10⁶one/mL.

6. The microfluidic colon cell study model for the study of the influence mechanism of novel coronaviruses on colon cells of claim 1, wherein said circulation path (114) is at the junction with said simulated external cavity (111) and said simulated internal cavity (113), and the direction of said exchange path is parallel to the surface of the barrier layer (112).

7. A microbubble research method for researching the influence mechanism of a novel coronavirus on lung cells, which is characterized by using the microfluidic colon cell research model for researching the influence mechanism of the novel coronavirus on colon cells as claimed in any one of claims 1 to 6.

8. The method for studying microvesicles for studying the mechanism of influence of a novel coronavirus on pulmonary cells according to claim 8, comprising the steps of:

1) taking the colon cell research model, circularly introducing blood simulation culture solution into the simulation outer cavity (111) through the circulation passage (114), and circularly flowing the blood simulation culture solution through the exchange hole connected with the blood simulation culture solution;

2) introducing air containing the novel coronavirus into the simulated outer cavity (111) in a circulating way through the circulating passage (114), and enabling the air to flow circularly through the exchange holes connected with the air;

3) after steps 1) and 2) are continued for a period of time, opening a liquid channel valve, collecting different cell secretion products and cells in different collecting pools (12), respectively, dismantling the colon cell research model, and taking out the barrier layer (112); an analytical study was conducted.

Technical Field

The invention belongs to the technical field of in-vitro simulation analysis, and particularly relates to a micro-fluidic colon cell research model and a micro-fluidic colon cell research method for researching the influence mechanism of a novel coronavirus on colon cells.

Background

The new coronavirus outbreak in 2019 arouses the general attention of the international society, and the world health organization has announced that the epidemic forms the emergent public health event of international attention in 1, 30 months in 2020. For human, the disease is always like a picture, the infectious disease is a large enemy of human, in 20 th century, smallpox captures the life of 3 hundred million people, Spanish virus outbreaks in 1918-1919-4 months cause 2500-4000 million deaths, influenza A H1N1 outbreaks in Mexico and California in 2009 in America lasts for more than one year, 28.45 million deaths are caused globally, Ebola virus spreads to a plurality of countries in short months, and the number of deaths reaches 7000.

There is a relatively high amount of ACE-2 expressed in colon cells and thus the novel coronavirus is recognized therewith, thus causing patients infected with the novel coronavirus to have symptoms of diarrhea. And may cause the production of a large amount of inflammatory factors, etc. At present, little is known about how the virus acts on colon cells, so that the construction of the research model is very meaningful.

The new coronavirus can be identified with ACE-2 on the surface of lung cells, and then enters the cells for proliferation. To study the effect of new coronavirus on human lung cells, long-term simulation studies were performed.

Although the animal model considers the influence of air pollutants on the organism from the whole, the animal model has high cost and long time consumption and is not easy to carry out quantitative research in vivo; in addition, due to species differences, animal models cannot represent physiological and pathological characteristics of human beings to some extent, and application of the animal models is limited.

In vitro cell models utilize cultured human lung cell lines for quantitative, high-throughput analysis, but because the in vivo cell environment cannot be realized, and information interaction and mechanical force action among various cells are lacked, the results are difficult to simulate the in vivo actual conditions.

Based on the above, there is a need for a micro-fluidic colon cell research model and a micro-fluidic colon cell research method for researching the influence mechanism of a novel coronavirus on colon cells, which can simulate the contraction and relaxation of colon caused by human respiration by rhythmic stretching of a vacuum pump while successfully simulating the qi-blood barrier, can construct a respiratory lung model in vitro, and has the advantages of low production cost, low requirement on production conditions, and convenient operation.

Disclosure of Invention

In order to solve the above problems, the present invention provides a microfluidic colon cell research model and method for researching the influence mechanism of novel coronavirus on colon cells, which solves the problems in the prior art.

The invention discloses a microfluidic colon cell research model for researching an influence mechanism of a novel coronavirus on colon cells, which comprises more than one culture mechanism, wherein one culture mechanism is selected as a control group, and other culture mechanisms are selected as experimental groups; the culture mechanism comprises a culture pond and more than one collecting pond; the inlet of the collecting tank is connected with the outlet of the culture tank through a liquid channel valve;

the culture pond comprises a simulated outer cavity, a barrier layer, a simulated inner cavity and a circulation passage; the barrier layer is arranged between the simulated outer cavity and the simulated inner cavity; the simulated outer cavity and the simulated inner cavity are respectively provided with the circulating passage; the barrier layer comprises a framework layer, a first simulation layer and a second simulation layer; the first simulation layer is a HUVEC cell layer proliferated on the first surface of the framework layer; the second simulation layer is an A549 cell layer proliferated on the second surface of the framework layer; the first simulation layer is connected with the simulation outer cavity, and the second simulation layer is connected with the simulation inner cavity.

Further, the inlet of the culture pond adopts a serpentine path. This reduces cell shear forces.

Furthermore, the framework layer is provided with holes, and the simulation layer covers the surface of the framework layer and simultaneously blocks the holes; or the framework layer adopts a semi-permeable membrane, and substances on two sides of the barrier layer can exchange substances with the simulation layer through the framework layer.

Preferably, the framework layer is made of polycarbonate.

Preferably, the density of A549 cells adopted by the second simulation layer is 1 x 10⁶Per mL; the cell density of human colon goblet cells HT29-CL.16E or HUVEC adopted by the first simulation layer is 1 × 10⁷one/mL.

Furthermore, the external shape of the simulated external cavity and the simulated external cavity is at least one of regular or irregular polygon, spindle, circle and ellipse.

Preferably, the simulated outer cavity and the simulated outer cavity are spindle-shaped in appearance.

Preferably, the circulation path is at the junction with the simulated external cavity and the simulated internal cavity, the direction of the exchange path being parallel to the barrier layer surface. The influence of the fluid flow on the cells can be reduced.

The invention also discloses a microbubble research method for researching the influence mechanism of the novel coronavirus on the lung cells, which comprises the step of adopting the microfluidic colon cell research model for researching the influence mechanism of the novel coronavirus on the colon cells.

Further, the microbubble research method for researching the influence mechanism of the novel coronavirus on the lung cells comprises the following steps:

1) taking the colon cell research model, circularly introducing blood simulation culture solution into the simulation outer cavity through the circulating passage, and circularly flowing the blood simulation culture solution through the exchange hole connected with the blood simulation culture solution;

2) introducing air containing the novel coronavirus into the simulated outer cavity in a circulating way through the circulating passage, and enabling the air to flow circularly through the exchange holes connected with the air;

3) after the steps 1) and 2) are continued for a period of time, opening a liquid channel valve, respectively collecting different cell secretion products and cells in different collecting pools, dismantling the colon cell research model, and taking out the barrier layer; an analytical study was conducted.

The invention has the beneficial effects that:

1. the simulation device has simple structure and low cost;

2. an A549 cell layer and a human colon goblet cell HT29-CL.16E or HUVEC cell layer are respectively arranged, so that the structure of colon capillaries is effectively simulated;

3. the simulation device is simple to operate;

4. the exchange cavity adopts a streamline spindle shape, and the factors of fluid mechanics are considered, so that uneven fluid flow is prevented.

Drawings

FIG. 1 is a schematic view of a structure of a culture apparatus according to the present invention;

FIG. 2 is a schematic of the present invention;

FIG. 3 is a schematic sectional view of the culture tank of the present invention;

FIG. 4 is a cross-sectional view of a barrier layer of the present invention;

FIG. 5 is a schematic view of a second structure of a culture tank of the present invention.

Detailed Description

The following examples are given to illustrate the technical embodiments of the present invention more clearly and should not be construed as limiting the scope of the present invention.

Example 1

As shown in attached figures 1-5, the invention discloses a microfluidic colon cell research model for researching the influence mechanism of a novel coronavirus on colon cells, which comprises more than one culture mechanism 1, wherein one culture mechanism 1 is selected as a control group, and the other culture mechanisms 1 are selected as experimental groups; the culture mechanism 1 comprises a culture pond 11 and more than one collecting pond 12; the inlet of the collecting tank 12 is connected with the outlet of the culture tank 11 through a liquid channel valve;

the culture pond 11 comprises a simulated outer cavity 111, a barrier layer 112, a simulated inner cavity 113 and a circulating passage 114; the barrier layer 112 is disposed between the simulated outer cavity 111 and the simulated inner cavity 113; the simulated outer cavity 111 and the simulated inner cavity 113 are respectively provided with the circulation passage 114; the barrier layer 112 comprises a skeleton layer 1121, a first simulation layer 1122, and a second simulation layer 1123; the first simulation layer 1122 is a layer of HUVEC cells proliferated on the first surface of the framework layer 1121; the second simulated layer 1123 is a layer of a549 cells proliferated on the second surface of the framework layer 1121; the first simulation layer 1122 is connected to the simulation outer chamber 111, and the second simulation layer 1123 is connected to the simulation inner chamber 113.

In this embodiment, the inlet of the culture pond 11 adopts a serpentine path. This reduces cell shear forces.

In this embodiment, the framework layer 1121 is provided with holes, and the simulation layer covers the surface of the framework layer 1121 and blocks the holes at the same time; or the framework layer 1121 adopts a semipermeable membrane, and materials on two sides of the barrier layer 112 can exchange materials with the simulation layer through the framework layer 1121.

Preferably, the framework layer 1121 is made of polycarbonate.

Preferably, the second mimic layer 1123 employs a549 cell density of 1 × 10⁶Per mL; the first simulated layer 1122 was designed with human colon goblet cells HT29-CL.16E or HUVEC at a cell density of 1X 10⁷one/mL.

In this embodiment, the external shapes of the simulated external cavity 111 and the simulated external cavity 111 are at least one of regular or irregular polygon, spindle shape, circle shape, and ellipse shape.

Preferably, the simulated outer cavity 111 and the simulated outer cavity 111 are spindle-shaped in shape.

Preferably, the circulation path 114 is at the junction with the simulated outer cavity 111 and the simulated inner cavity 113, and the direction of the exchange path is parallel to the surface of the barrier layer 112. The influence of the fluid flow on the cells can be reduced.

The invention also discloses a microbubble research method for researching the influence mechanism of the novel coronavirus on the lung cells, which comprises the step of adopting the microfluidic colon cell research model for researching the influence mechanism of the novel coronavirus on the colon cells.

In this embodiment, the method for studying microvesicles for studying the mechanism of influence of the novel coronavirus on lung cells comprises the following steps:

1) taking the colon cell research model, circularly introducing blood simulation culture solution into the simulation outer cavity 111 through the circulation passage 114, and circularly flowing the blood simulation culture solution through the exchange holes connected with the blood simulation culture solution;

2) air containing the novel coronavirus is circularly introduced into the simulated outer cavity 111 through the circulating passage 114, and the air circularly flows through the exchange holes connected with the air;

3) after steps 1) and 2) are continued for a period of time, opening a liquid channel valve, collecting different cell secretion products and cells in different collecting tanks 12 respectively, dismantling the colon cell research model, and taking out the barrier layer 112; an analytical study was conducted.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.